Repurposing Study of 4-Acyl-1-phenylaminocarbonyl-2-substituted-piperazine Derivatives as Potential Anticancer Agents—In Vitro Evaluation against Breast Cancer Cells

Breast cancer is the most common type of cancer in women. Although current treatments can increase patient survival, they are rarely curative when the disease is advanced (metastasis). Therefore, there is an urgent need to develop new cytotoxic drugs with a high selectivity toward cancer cells. Since repurposing approved drugs for cancer therapy has been a successful strategy in recent years, in this study, we screened a library of antiviral piperazine-derived compounds as anticancer agents. The compounds included a piperazine ring and aryl urea functions, which are privileged structures present in several anti-breast cancer drugs. The selective cytotoxic activity of a set of thirty-four 4-acyl-2-substituted piperazine urea derivatives against MCF7 breast cancer cells and MCF 10A normal breast cells was determined. Compounds 31, 32, 35, and 37 showed high selective anticancer activity against breast cancer cells and were also tested against another common type of cancer, non-small cell lung cancer (A549 lung cancer cells versus MRC-5 lung normal cells). Compounds 35 and 37 also showed selectivity against lung cancer cells. These results suggest that compounds 35 and 37 may be promising hit compounds for the development of new anticancer agents.


Introduction
With over 2.3 million new cases estimated, breast cancer was the most prevalent cancer diagnosed worldwide at the end of 2020 [1].By 2040, the burden of breast cancer is estimated to increase to more than 3 million new cases and 1 million deaths every year due to the growth and aging of the population [2].Most deaths are caused by metastasis, that is, when cancer cells have spread to other tissues and organs [3].Although pharmacotherapy, the main form of treatment for patients with metastases, can prolong patient survival, it is not enough to cure the cancer in most cases.Most current anticancer drugs have narrow selectivity toward cancer cells, and they also damage healthy cells, especially those with a high proliferative rate, such as cells of the immune system.Therefore, anticancer drugs are usually used in doses that are tolerable but not enough to kill all cancer cells, and the disease progresses to death.New drugs that improve the efficacy of current antitumor therapies for breast cancer are urgently needed.Medicinal chemists play an important role in the discovery of anticancer agents through the design, optimization, and development of new chemical compounds [4][5][6][7].
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 2 of 19 survival, it is not enough to cure the cancer in most cases.Most current anticancer drugs have narrow selectivity toward cancer cells, and they also damage healthy cells, especially those with a high proliferative rate, such as cells of the immune system.Therefore, anticancer drugs are usually used in doses that are tolerable but not enough to kill all cancer cells, and the disease progresses to death.New drugs that improve the efficacy of current antitumor therapies for breast cancer are urgently needed.Medicinal chemists play an important role in the discovery of anticancer agents through the design, optimization, and development of new chemical compounds [4][5][6][7].
The piperazine scaffold represents a very important class of bioactive N-heterocyclic compounds due to their pharmacological versatility being widely used in research for the development of new molecules for diverse therapeutic targets.It has been reported that a wide range of biological applications of molecules containing piperazine are an essential subunit of the structural frame.These include antibacterials and antifungals [8][9][10][11][12], antiviral [13][14][15][16], antipsychotic [17], antihypertensive [18], and antioxidant [19], among others.Many currently notable anticancer drugs, such as imatinib (STI571) [20], dasatinib (BMS-354825) [21], bosutinib (SKI-606) [22], danusertib (PHA-739358) [23], and VX-680 [24], contain a piperazine ring as part of their molecular structure (Figure 1).In the literature, novel piperazine derivatives or skeletons containing piperazine have been described as antiproliferative agents against breast cancer (Figure 2) [25][26][27].Hybrid compounds containing pharmacophores with 4-piperazinyl-quinoline-isatin derivatives (1) exhibited cytotoxicity against two human breast cancer cell lines (MDA-MB468 and MCF7), with IC50 values in the range of 10.34-66.78µM [25].Three of the compounds tested showed two-to four-fold lower cytotoxic effects in noncancerous breast cell lines (184B5 and MCF 10A).Thiouracil amide derivatives bearing a piperazine ring (2) were synthesized and screened for cytotoxic activity in MCF7 cells, with IC50 values in the range of 18.23 to 100 µM [26].The most active compounds, those with tolyl or halophenyl group alkylating the piperazine ring, were two to three times less cytotoxic against normal MCF 10A cells than cancer cells.Also, small molecules such as 1-(4-substitutedbenzoyl)-4-(4chlorobenzhydryl) piperazine derivatives (3) with different groups at the aryl amide function showed cytotoxic activity against breast cancer cells (MCF7, BT20, T47D, and CAMA-1) and other types of cancer cell lines [27].These compounds were also evaluated in normal breast epithelial cells (MCF-12A).The IC50 values were in the range of 0.31-120.52µM against breast cancer cell lines and 6.6-299.66µM against normal breast cells.In the literature, novel piperazine derivatives or skeletons containing piperazine have been described as antiproliferative agents against breast cancer (Figure 2) [25][26][27].Hybrid compounds containing pharmacophores with 4-piperazinyl-quinoline-isatin derivatives (1) exhibited cytotoxicity against two human breast cancer cell lines (MDA-MB468 and MCF7), with IC 50 values in the range of 10.34-66.78µM [25].Three of the compounds tested showed two-to four-fold lower cytotoxic effects in noncancerous breast cell lines (184B5 and MCF 10A).Thiouracil amide derivatives bearing a piperazine ring (2) were synthesized and screened for cytotoxic activity in MCF7 cells, with IC 50 values in the range of 18.23 to 100 µM [26].The most active compounds, those with tolyl or halophenyl group alkylating the piperazine ring, were two to three times less cytotoxic against normal MCF 10A cells than cancer cells.Also, small molecules such as 1-(4-substitutedbenzoyl)-4-(4-chlorobenzhydryl) piperazine derivatives (3) with different groups at the aryl amide function showed cytotoxic activity against breast cancer cells (MCF7, BT20, T47D, and CAMA-1) and other types of cancer cell lines [27].These compounds were also evaluated in normal breast epithelial cells (MCF-12A).The IC 50 values were in the range of 0.31-120.52µM against breast cancer cell lines and 6.6-299.66µM against normal breast cells.
The urea function also constitutes a relevant moiety in anticancer agents.Biaryl-ureabased kinase inhibitors have been approved in anticancer therapy, for example, sorafenib (4) and its analog, regorafenib (5).The development of novel biaryl-urea-based small molecules as potential kinase inhibitors has been increasingly highlighted with the clinical success of these compounds [28], such as compound 6, a multikinase inhibitor that showed significant anticancer activity against diverse cancer cells [29] (Figure 3).The urea function also constitutes a relevant moiety in anticancer agents.Biaryl-ureabased kinase inhibitors have been approved in anticancer therapy, for example, sorafenib (4) and its analog, regorafenib (5).The development of novel biaryl-urea-based small molecules as potential kinase inhibitors has been increasingly highlighted with the clinical success of these compounds [28], such as compound 6, a multikinase inhibitor that showed significant anticancer activity against diverse cancer cells [29] (Figure 3).Drug repurposing or repositioning means establishing new medical uses for alreadyknown drugs.Although this is not a new strategy, it has become more popular in recent years, reaching around 25% of the annual pharmaceutical industry revenue.In the past decades, several drugs that were originally approved for indications other than cancer treatment have shown antitumoral effects [30,31].
The objective pursued in this study was to screen a library of antiviral piperazinederived compounds for their possible repositioning as anticancer agents since piperazine ring and aryl urea functions are privileged structures present in anticancer drugs.

Results and Discussion
In this work, we report the biological evaluation of a set of 34 compounds 4-N-acyl-1-phenylamino(thio)carbonyl-2-substituted piperazine derivatives (previously described as effective antiadenovirus agents) [15] against breast cancer cells.Figure 4 shows the general scaffold of our compounds that consists of a piperazine ring as a central core with a phenylamino(thio)carbonyl group at nitrogen 1 and different acyl groups at nitrogen 4.  The urea function also constitutes a relevant moiety in anticancer agents.Biaryl-ureabased kinase inhibitors have been approved in anticancer therapy, for example, sorafenib (4) and its analog, regorafenib (5).The development of novel biaryl-urea-based small molecules as potential kinase inhibitors has been increasingly highlighted with the clinical success of these compounds [28], such as compound 6, a multikinase inhibitor that showed significant anticancer activity against diverse cancer cells [29] (Figure 3).Drug repurposing or repositioning means establishing new medical uses for alreadyknown drugs.Although this is not a new strategy, it has become more popular in recent years, reaching around 25% of the annual pharmaceutical industry revenue.In the past decades, several drugs that were originally approved for indications other than cancer treatment have shown antitumoral effects [30,31].
The objective pursued in this study was to screen a library of antiviral piperazinederived compounds for their possible repositioning as anticancer agents since piperazine ring and aryl urea functions are privileged structures present in anticancer drugs.

Results and Discussion
In this work, we report the biological evaluation of a set of 34 compounds 4-N-acyl-1-phenylamino(thio)carbonyl-2-substituted piperazine derivatives (previously described as effective antiadenovirus agents) [15] against breast cancer cells.Figure 4 shows the general scaffold of our compounds that consists of a piperazine ring as a central core with a phenylamino(thio)carbonyl group at nitrogen 1 and different acyl groups at nitrogen 4. Drug repurposing or repositioning means establishing new medical uses for alreadyknown drugs.Although this is not a new strategy, it has become more popular in recent years, reaching around 25% of the annual pharmaceutical industry revenue.In the past decades, several drugs that were originally approved for indications other than cancer treatment have shown antitumoral effects [30,31].
The objective pursued in this study was to screen a library of antiviral piperazinederived compounds for their possible repositioning as anticancer agents since piperazine ring and aryl urea functions are privileged structures present in anticancer drugs.

Results and Discussion
In this work, we report the biological evaluation of a set of 34 compounds 4-N-acyl-1-phenylamino(thio)carbonyl-2-substituted piperazine derivatives (previously described as effective antiadenovirus agents) [15] against breast cancer cells.Figure 4 shows the general scaffold of our compounds that consists of a piperazine ring as a central core with a phenylamino(thio)carbonyl group at nitrogen 1 and different acyl groups at nitrogen 4. Our general skeleton has three points of structural variation: the substituent on the piperazine ring (R 1 : methyl or phenyl), the acyl group at N-4 (R 2 : pivaloyl, benzoyl, tertbutoxycarbonyl, or benzofuran-2-carbonyl) and the different substituents at the phenyl Our general skeleton has three points of structural variation: the substituent on the piperazine ring (R 1 : methyl or phenyl), the acyl group at N-4 (R 2 : pivaloyl, benzoyl, tertbutoxycarbonyl, or benzofuran-2-carbonyl) and the different substituents at the phenyl ring in the urea function (electron-withdrawing or electron-releasing groups).
The collection of compounds 7-39 is shown in Table 1.
The collection of compounds 7-39 is shown in Table 1.To evaluate the cytotoxicity of our compounds against cancer cells, they were evaluated in vitro against MCF7 human breast cancer cells.To examine the selectivity of our compounds, they were also studied against MCF 10A human normal cells.Both cell lines were exposed to different concentrations of our compounds for 72 h before measuring cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (for conditions of the MTT assay, see the Experimental Part, Section 3).Docetaxel, an anticancer drug used in clinics [32], was tested under the same experimental conditions.Data were obtained from at least two independent experiments and were expressed as means ± standard error of means (SEM).The half-maximal inhibitory concentration (IC 50 ) value of each compound was calculated, and the results are collected in Table 2. To evaluate the selective activity of our compounds, the selectivity index (SI) was also calculated.The curves of cell viability of MCF7 cancer cells and MCF 10A normal cells after the treatment with our compounds are shown (Figures 5-9  To evaluate the cytotoxicity of our compounds against cancer cells, they were evaluated in vitro against MCF7 human breast cancer cells.To examine the selectivity of our compounds, they were also studied against MCF 10A human normal cells.Both cell lines were exposed to different concentrations of our compounds for 72 h before measuring cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (for conditions of the MTT assay, see the Experimental Part, Section 3).Docetaxel, an anticancer drug used in clinics [32], was tested under the same experimental conditions.Data were obtained from at least two independent experiments and were expressed as means ± standard error of means (SEM).The half-maximal inhibitory concentration (IC50) value of each compound was calculated, and the results are collected in Table 2. To evaluate the selective activity of our compounds, the selectivity index (SI) was also calculated.The curves of cell viability of MCF7 cancer cells and MCF 10A normal cells after the treatment with our compounds are shown (Figures 5-9 and S1-S4).a Data averaged from at least two independent experiments are expressed as mean ± standard error of the mean (SEM).b Selectivity index was calculated by dividing the IC 50 value obtained in the nonmalignant cell line by that in the cancer cell line.

Structure Activity Relationship
In order to obtain structural information about which organic function contributes to the biological activity, firstly, the methyl piperazine ring was selected as the central core, and chemical diversity was focused on N-4 by the presence of three different acyl groups (pivaloyl, benzoyl, and tert-butoxycarbonyl). NO 2 and OMe (at para position of the phenyl ring in the urea) were selected as prototypes of groups with both electronic properties mentioned above.See Table 2 for the biological data mentioned in the discussion.
Compounds 7, 10, and 12 (pivaloyl, benzoyl, and tert-butoxycarbonyl derivatives, respectively) with a 4-NO 2 group at the phenyl ring in the urea function were more active than their analogs with a 4-OMe group (8, 11, and 13 respectively), which showed higher IC 50 values against MCF7 cancer cells (Figures 5 and S1).Among the NO 2 derivatives, 10 (with an aromatic amide at N-4) gave the lowest IC 50 (26.7 µM).An electron-withdrawing group is important for the activity in this type of compound.However, compounds 7, 10, and 12 proved to be less selective against cancer cells than their analogs.
Two modifications were present in the analogs that allowed us to study their effect on anticancer activity.
Due to the benzofuran ring being an important privileged structure for anticancer activity compounds [33], the 2-methylpiperazines (same central core) having a 2-benzofuran-2-carbonyl at N-4 were also evaluated (Figures 6 and S2).A set of different electronwithdrawing groups in the phenyl ring in the urea function were considered mainly in para position but also in ortho position.In general terms, the benzofuran-2-carbonyl derivatives with an electron-withdrawing group at para position were more active against the cancer cell line than those with other acyl groups at N-4, giving lower IC 50 values (compound 18, 4-NO 2 , 12.1 µM; compound 19, 4-Cl, 30.5 µM; compound 20, 4-CF 3 , 31.7 µM; and compound 21, 4-CN, 19.4 µM).Related to the presence of the group in ortho position, it did not optimize the activity.The 2-NO 2 22, 2-OCH 3 23, and 2-Br 24 derivatives showed similar IC 50 (25.3-31.4µM).Compound 25, with an unsubstituted phenyl ring in the urea, gave the highest IC 50 value (48.5 µM).From these screenings, it can be concluded that the benzofurane ring and an electron-withdrawing group at para position in the phenyl of the urea are structural features linked to both nitrogens of the 2-methyl piperazine core that contribute to increased anticancer activity.
It is important to note that the selective anticancer effects of the compounds remain an issue to be addressed.For the most active derivatives, 9, 18, and 21, with IC 50 < 20 µM, the highest SI was 4.2 (compound 9).Compounds 19 and 20 showed higher SI, being approximately eight times more toxic to cancer cells than normal cells; however, these compounds display selective anticancer activity at concentrations within a narrow range (Figure 6).
At this point, we were focused on increasing the anticancer activity and on reducing the toxicity.Since the replacement of the 2-methyl piperazine core by 2-phenyl piperazine led to more active and noncytotoxic antiviral compounds [15], this backbone was also present in the tested compounds.tert-Butoxycarbonyl and benzofuran-2-carbonyl were the two acyl moieties at N-4.
Related to the selective profile, the SI values for these 2-phenyl-piperazine derivatives also increased, highlighting two compounds (31 and 32) with 16.6 and 12.6 values, respectively.
The last structural consideration was the presence of the benzofuran-2-carbonyl group at N-4, keeping electron-withdrawing groups at the urea.Compounds with 4-NO 2 (35), 4-Cl (36), 4-CN (37), 2-Cl,5-CF 3 (38), and 2-NO 2 (39) were less cytotoxic against MCF7 cancer cells (Figures 8 and S4) than their Boc analogs.The benzofuran-2-carbonyl analogs showed higher IC 50 (23.0µM, 41.3 µM, 57.1 µM, 91.9 µM, and 225.9 µM, respectively).In general terms, the Boc 2-phenyl derivatives presented a better cytotoxic activity profile (lower IC 50 ) than the corresponding benzofuran-2-carbonyl analogs (26  The comparison of both types of piperazine core showed that the 2-phenylpiperazine skeleton generated compounds with higher SI, showing less cytotoxicity against normal cells than the 2-methyl derivatives. From this collection (Table 1), based on the experimental data described, five Boc 2phenyl derivatives with IC 50 < 20 µM were chosen as potential agents against breast cancer: 26, 27, 28, 31, and 32.They were submitted for in silico evaluation of their physicochemical and pharmacokinetic properties.31 and 32 resulted the most promising compounds, with better selective anticancer activity compared with docetaxel (Table 2).Docetaxel is a common anticancer agent used in the treatment of solid tumors, including breast cancer.However, this drug induces numerous adverse effects because it also affects normal tissues.Under our experimental conditions, MCF 10A normal cells were more sensitive to docetaxel than MCF7 cancer cells (SI < 1).Similar results have been reported by other authors [34,35].In contrast, compounds 31 and 32 were more cytotoxic on MCF7 cancer cells than MCF 10A normal cells.
Having in mind the fact that the presence of the benzofuran-2-carbonyl group at N-4 in the 2-methyl piperazine core gave better activity data than the Boc group (lower IC 50 values were observed), we decided to preserve both characteristics and replace the aryl group at the urea function.We prepared a conjugate of these two-membered scaffolds with adamantane moiety, also employing the urea function as a linker connecting the molecular fragments.We chose this polycyclic cage because it has been described as a privileged structure in promising anticancer agents [36][37][38], also present in hybrids with other active compounds such as colchicine (an ester group was the linker connector), revealing high cytotoxicity to cancer cells [39].
The synthetic methodology was a brief and high-yielded route, previously described, that consists of two reactions (Scheme 1) [15,16].The first step involved the introduction of the benzofuran-2-carbonyl group through a chemoselective N-acylation reaction of 2methyl piperazine with the appropriate acyl chloride reactive providing the introduction of the amide function at the less hindered nitrogen (40).From this monoacyl derivative, the urea group was introduced at the other nitrogen by reaction with adamantyl isocyanate.Compound 41 was obtained in high yield.Spectral (nuclear magnetic resonance (NMR) and mass spectrometry) and analytical data of 41 showed full agreement with the proposed structure (see Experimental Part, Section 3, Figures S5 and S6, Supplementary Information).with adamantane moiety, also employing the urea function as a linker connecting the molecular fragments.We chose this polycyclic cage because it has been described as a privileged structure in promising anticancer agents [36][37][38], also present in hybrids with other active compounds such as colchicine (an ester group was the linker connector), revealing high cytotoxicity to cancer cells [39].
The synthetic methodology was a brief and high-yielded route, previously described, that consists of two reactions (Scheme 1) [15,16].The first step involved the introduction of the benzofuran-2-carbonyl group through a chemoselective N-acylation reaction of 2methyl piperazine with the appropriate acyl chloride reactive providing the introduction of the amide function at the less hindered nitrogen (40).From this monoacyl derivative, the urea group was introduced at the other nitrogen by reaction with adamantyl isocyanate.Compound 41 was obtained in high yield.Spectral (nuclear magnetic resonance (NMR) and mass spectrometry) and analytical data of 41 showed full agreement with the proposed structure (see Experimental Part, Section 3, Figures S5 and S6, Supplementary Information).Under the same experimental conditions, compound 41 also showed cytotoxicity against the MCF7 breast cancer cell line and selective anticancer activity (Figure 9).MCF7 cancer cells were ~5 times more sensitive to compound 41 than MCF 10A normal cells, with IC50 values (mean ± SEM; µM) of 17.7 ± 5.0 and 102.7 ± 66.9, respectively.SI (mean ± SEM) was 5.1 ± 2.3.Compound 41 was also selected for in silico evaluation of drug-likeness properties.
In summary, among the thirty-four compounds examined, seven compounds showed high cytotoxic activity against breast cancer cells, with IC50 ranging from 12.1 to 19.5 µM, and two showed IC50 values of <10 µM (2.7 and 9.6 µM).Four compounds (31,32,35,37) were >10 times more cytotoxic to cancer cells than to normal cells, being the most selective anticancer compounds.

Evaluation of the Selective Cytotoxic Activity of 4-N-acyl-1-phenylamino(thio)carbonyl-2-substituted Piperazine Derivatives against Nonmalignant Cells and Lung Cancer Cells
Benzofurane scaffold, piperazine ring, and urea function are relevant privileged structures present not only in anticancer drugs against breast cancer but also against other types of cancer, such as lung cancer, hepatocarcinoma, and gastric cancer [20,28,[40][41][42][43]].To identify the potential broad-spectrum anticancer activity of our piperazine derivatives, we tested the most selective compounds in non-small cell lung cancer, the second most common cancer worldwide after breast cancer and the leading cause of cancer-related deaths [1].MRC-5 normal lung cells and A549 lung cancer cells were exposed to compounds 31, 32, 35, 37, and cisplatin (a common drug used to treat lung cancer) for 72 h, and cell viability was measured using the MTT assay (Figure 10 and Table 3).Compounds 35 and 37 also had high selectivity for A549 cancer cells.While compound 35 showed Under the same experimental conditions, compound 41 also showed cytotoxicity against the MCF7 breast cancer cell line and selective anticancer activity (Figure 9).MCF7 cancer cells were ~5 times more sensitive to compound 41 than MCF 10A normal cells, with IC 50 values (mean ± SEM; µM) of 17.7 ± 5.0 and 102.7 ± 66.9, respectively.SI (mean ± SEM) was 5.1 ± 2.3.Compound 41 was also selected for in silico evaluation of drug-likeness properties.
In summary, among the thirty-four compounds examined, seven compounds showed high cytotoxic activity against breast cancer cells, with IC 50 ranging from 12.1 to 19.5 µM, and two showed IC 50 values of <10 µM (2.7 and 9.6 µM).Four compounds (31,32,35,37) were >10 times more cytotoxic to cancer cells than to normal cells, being the most selective anticancer compounds.

Evaluation of the Selective Cytotoxic Activity of 4-N-acyl-1-phenylamino(thio)carbonyl-2-substituted Piperazine Derivatives against Nonmalignant Cells and Lung Cancer Cells
Benzofurane scaffold, piperazine ring, and urea function are relevant privileged structures present not only in anticancer drugs against breast cancer but also against other types of cancer, such as lung cancer, hepatocarcinoma, and gastric cancer [20,28,[40][41][42][43]].To identify the potential broad-spectrum anticancer activity of our piperazine derivatives, we tested the most selective compounds in non-small cell lung cancer, the second most common cancer worldwide after breast cancer and the leading cause of cancer-related deaths [1].MRC-5 normal lung cells and A549 lung cancer cells were exposed to compounds 31, 32, 35, 37, and cisplatin (a common drug used to treat lung cancer) for 72 h, and cell viability was measured using the MTT assay (Figure 10 and Table 3).Compounds 35 and 37 also had high selectivity for A549 cancer cells.While compound 35 showed similar IC 50 values in A549 and MCF7 cells (25.6 and 23.0 µM, respectively), compound 37 was more cytotoxic against A549 than MCF7 cells (18.7 and 57.1 µM, respectively).It should be mentioned that both compounds showed a slightly better selective profile against lung cancer cells than the anticancer drug cisplatin.On the other hand, compounds 31 and 32 showed lower IC 50 values against A549 than compounds 35 and 37, but they were also very toxic for MRC-5 normal cells, showing lower selective profiles.similar IC50 values in A549 and MCF7 cells (25.6 and 23.0 µM, respectively), compound 37 was more cytotoxic against A549 than MCF7 cells (18.7 and 57.1 µM, respectively).It should be mentioned that both compounds showed a slightly better selective profile against lung cancer cells than the anticancer drug cisplatin.On the other hand, compounds 31 and 32 showed lower IC50 values against A549 than compounds 35 and 37, but they were also very toxic for MRC-5 normal cells, showing lower selective profiles.a Data averaged from at least two independent experiments are expressed as mean ± standard error of the mean (SEM).b Selectivity index was calculated by dividing the IC50 value obtained in the nonmalignant cell line by that in the cancer cell line.
One key limitation of most anticancer drugs is that they not only target cancer cells but also affect nonmalignant cells with high division rates.This can induce numerous adverse effects, such as severe nausea and vomiting, hair loss, diarrhea, mouth sores, anemia, erythematous eruptions, etc.For example, skin cells, which are constantly renewing themselves, are often damaged by antimetabolites or alkylating agents.This can lead to painful erythematous eruptions [44].Because MCF 10A and MRC-5 are normal cells with  a Data averaged from at least two independent experiments are expressed as mean ± standard error of the mean (SEM).b Selectivity index was calculated by dividing the IC 50 value obtained in the nonmalignant cell line by that in the cancer cell line.
One key limitation of most anticancer drugs is that they not only target cancer cells but also affect nonmalignant cells with high division rates.This can induce numerous adverse effects, such as severe nausea and vomiting, hair loss, diarrhea, mouth sores, anemia, erythematous eruptions, etc.For example, skin cells, which are constantly renewing themselves, are often damaged by antimetabolites or alkylating agents.This can lead to painful erythematous eruptions [44].Because MCF 10A and MRC-5 are normal cells with a low proliferation rate, we decided to test the most selective compounds on nonmalignant HaCaT cells.These cells are derived from normal adult tissue and have a division rate similar to that of cancer cells.A549 cells and HaCaT cells were exposed to various concentrations of 31, 32, 35, 37, or gemcitabine (a common antimetabolite used to treat cancer) for 72 h.Cell viability was determined with the resazurin assay.Subsequently, the cells were allowed to grow in a drug-free medium for another 72 h.This was performed to test their survival and proliferation ability after the removal of the compounds.Cell viability was again measured with the resazurin assay.The results are represented in Figure 11 and Table 4. Compounds 32, 35, and 37 showed similar IC 50 values for A549 to those obtained with the MTT assay.Compounds 31, 35, and 37 showed selective anticancer activity, although the selectivity profile was lower than against the normal cell lines previously described.A549 cancer cells were approximately two times more sensitive to compounds 31, 35, and 37 than nonmalignant HaCaT cells.It should be mentioned that cells exposed to high concentrations of compounds above 100 µM were unable to recover after treatment.
a low proliferation rate, we decided to test the most selective compounds on nonmalignant HaCaT cells.These cells are derived from normal adult tissue and have a division rate similar to that of cancer cells.A549 cells and HaCaT cells were exposed to various concentrations of 31, 32, 35, 37, or gemcitabine (a common antimetabolite used to treat cancer) for 72 h.Cell viability was determined with the resazurin assay.Subsequently, the cells were allowed to grow in a drug-free medium for another 72 h.This was performed to test their survival and proliferation ability after the removal of the compounds.Cell viability was again measured with the resazurin assay.The results are represented in Figure 11 and Table 4. Compounds 32, 35, and 37 showed similar IC50 values for A549 to those obtained with the MTT assay.Compounds 31, 35, and 37 showed selective anticancer activity, although the selectivity profile was lower than against the normal cell lines previously described.A549 cancer cells were approximately two times more sensitive to compounds 31, 35, and 37 than nonmalignant HaCaT cells.It should be mentioned that cells exposed to high concentrations of compounds above 100 µM were unable to recover after treatment.
Figure 11.Evaluation of cytotoxic activity of compounds 31, 32, 35, 37, and gemcitabine on human nonmalignant cells (HaCaT) and human cancer cells (A549).Cells were exposed to several concentrations of compounds for 72 h ("72 h T", continuous lines), and cell viability was determined with the resazurin assay.Subsequently, the cells were washed and were allowed to grow in drug-free Cells were exposed to several concentrations of compounds for 72 h ("72 h T", continuous lines), and cell viability was determined with the resazurin assay.Subsequently, the cells were washed and were allowed to grow in drug-free medium for another 72 h ("72 h T + 72 h R", discontinue lines).Cell viability was again measured with the resazurin assay.Data represent mean ± SEM from at least three independent experiments.

In Silico Evaluation of Physicochemical and Pharmacokinetic Properties of Selected Compounds
In the drug development process, most new drug candidates fail in clinical trials due to their reduced ADME (absorption, distribution, metabolism, and excretion) properties.In silico ADME screens provide a good starting point for selecting the most promising candidates for development and rejecting those with a low chance of success [45].Selected compounds (26-28, 31, 32, 35, 37, and 41) were tested for drug likeness by Lipinski's rule of five [46] through the SwissADME online tool (available from URL: http://http://www.swissadme.ch,accessed on 29 November 2023) [47] (Table S1, Supplementary Information).All the selected compounds mirrored Lipinski's rule.Additionally, compounds with polar surface area (PSA) lower than 140 A 2 and 10 or fewer rotatable bonds should exhibit high oral bioavailability [48].Polar surface area is a factor involved in the calculation of percentage absorption (%ABS) being inversely proportional to %ABS by the following equation (%ABS = 109 − (0.345 TPSA) [47], with consequent predicted percentage oral absorption from 70.49 to 87.65% indicating good permeability and transport via biological membranes [49,50].
Regarding the ADME properties of the compounds, we decided to analyze some key features that are relevant to the druggability profile of a target compound, such as absorption and distribution, using the Pre-ADMET software, from BMDRC, Seoul, Republic of Korea (available from URL: http://preadmet.bmdrc.org,accessed 29 November 2023) [51,52].Human intestinal absorption (HIA), Caco-2 cell permeability, skin permeability, bloodbrain barrier penetration (BBB), and plasma protein binding (PPB) were predicted using this program.The outcomes of the predicted ADME parameters are shown in Table S2, Supplementary Information.
All the compounds were predicted with a good intestinal absorption above 96% (wellabsorbed compounds: 70-100%ABS) and medium cell permeability in the Caco-2 model, with values from 21.80 to 45.10 nm/s (normal range 4-70 nm/s).
BBB is an important predictor for central nervous system (CNS) drug discovery.The obtained results showed two types of CNS access: five compounds (26, 28, 35, 37, and 41) displayed low CNS absorption, with values < 0.1, and three compounds (27, 31, and 32) gave values ranging from 0.1 to 2.0, exhibiting medium CNS absorption.31 and 32, those with Cl and CF 3 di-substitution patterns (Log P < 4.6), were the most highly absorbed in CNS [49].
Results also showed that six compounds were weakly bound to plasma protein, displaying PPB values of <90%, although three of them (31, 32, and 41) gave 89.0-89.44 values near enough to be considered strongly bounded.Compounds 35 and 37 displayed values of >90%.
In general terms, these selected compounds presented predicted physicochemical and pharmacokinetic properties that support their consideration as promising hits for the development of a novel class of anticancer agents.

Cell Lines
The human A549 lung adenocarcinoma cells and the human lung fibroblasts MRC-5 were purchased from the European Collection of Cell Cultures (Salisbury, UK).The human MCF7 breast adenocarcinoma and human MCF 10A breast epithelial cell lines were generously provided by Dr. D. Ruano and Dr. P. Daza (University of Seville), who obtained the cells from the American Type Culture Collection (ATCC, Manassas, VA, USA) [53].The human keratinocytes HaCaT cells [54] were purchased from the Cell Line Services (CLS, Hamburg, Germany).

Cell Viability Assays
Exponentially growing cells were seeded into 96-well plates, and drugs were added 24 h later.Each compound was dissolved with DMSO (Panreac Applichem) to prepare a stock solution (100 mM).The working solutions of specific concentrations were prepared via the dilution of the stock solutions in the culture medium and were immediately used to treat the cells.The remaining stock solutions were aliquoted and frozen at −40 • C. We used different aliquots in each independent experiment to avoid the freeze-thaw cycles.Cells were exposed to the drugs for 72 h.After these 72 h, cell viability was determined with the MTT assay or the resazurin assay.These assays are colorimetric techniques that are widely used to determine cell viability.
The MTT assay is based on the capability of viable cells to transform the MTT salt into a purple formazan dye.After an incubation period of the cells with the MTT and a solubilization step, the quantity of the colored product is measured with a plate reading spectrophotometer.Dead cells are metabolically inactive and cannot produce the colored product.The quantity of formazan produced is presumably proportional to the number of viable cells [55].After seeding for 24 h, cells were treated with the tested drugs for 72 h.Then, medium was removed, and 125 µL of MTT (1 mg/mL in medium) was added to each well for 3-4 h.Subsequently, 80 µL of 20% sodium dodecyl sulfate (Panreac Applichem) in 20 mM hydrochloric acid (Panreac Applichem) was added to dissolve the insoluble purple formazan product, plates were incubated for 10 h at 37 • C, and optical densities were measured at 540 nm on a multiwell plate spectrophotometer reader.
The resazurin assay measures the ability of viable cells to transform the blue compound resazurin into the pink compound resorufin.There is a direct correlation between the reduction in resazurin and the number of viable cells.After seeding for 24 h, cells were exposed to several concentrations of compounds.After a 72 h treatment period, the cells were washed once with a phosphate-buffered saline (PBS, biowest), and 150 µL of resazurin (20 µg/mL in the medium) was added to each well.The plates were then incubated for 4-5 h at 37 • C, 5% CO 2 , and optical densities were measured at 540 nm and 620 nm using a multiwell plate spectrophotometer reader (Imark Bio Rad Laboratories Inc., Hercules, CA, USA).Subsequently, cells were washed once with PBS and allowed to grow for an additional 72 h in drug-free medium to allow them to recuperate from possible damage induced by the tested compounds.After the treatment period, cell viability was measured following the same protocol described above.
In both assays, cell viability was expressed as percentage in relation to controls.All data were averaged from at least two independent experiments and were expressed as mean ± standard error of the mean (SEM).Selectivity indices (SI) were calculated by dividing the IC 50 values in the nonmalignant cells by those in the cancer cells [56].

General Methods
All reagents, solvents, and starting materials were obtained from commercial suppliers and were used without further purification.The crude reaction mixtures were concentrated under reduced pressure by removing the organic solvents in a rotary evaporator.Reactions were monitored by thin layer chromatography (TLC) using Kieselgel 60 F 254 (Merck, Rahway, NJ, USA) plates and UV detector for visualization.Flash column chromatography was performed on Silica Gel 60 (Merck).All reported yields were of purified products.Melting points were obtained on a Stuart Melting Point Apparatus SMP 10 and were uncorrected.Mass spectra were recorded on an Orbitrap Elite (Thermo Scientific, Whaltman, MA, USA), a hybrid ion trap-orbitrap mass spectrometer capable of acquiring a resolution higher than 240,000, with ESI, HESI, APCI, and nanoESI ionization sources.NMR spectra were recorded at 25 • C on a Bruker AV500 spectrometer at 500 MHz for 1 H and 125 MHz for 13 C.The chemical shifts (δ) reported are given in parts per million (ppm) on the δ scale relative to TMS, and the coupling constants (J) are in hertz (Hz). 1 H chemical shift values (δ) are referenced to the residual nondeuterated components of the NMR solvents (δ = 7.26 ppm for CDCl 3 ).The 13 C chemical shifts (δ) are referenced to deuterated solvent (central peak, δ = 77.16ppm) as the internal standard.The spin multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), or br s (broad singlet).The purity of final compounds was evaluated by elemental analysis (C, H, and N).The purity of all the final compounds was confirmed to be ≥95% by combustion.

Conclusions
In this study, a collection of 34 compounds of 4-acyl-1-phenylamino(thio)carbonyl-2substituted piperazine derivatives were evaluated against breast cancer cells, searching for small molecules that can be considered promising anticancer agents.The structural requirements that improve the cytotoxic and selectivity profiles were the central core of 2-pheny-piperazine possessing a Boc group at N-4 and an aryl urea function at N-1 with electron-withdrawing substituents at para position of the phenyl ring.
When the 2-methyl-piperazine ring was present, the best activity was observed in those compounds with a benzofuran-2-carbonyl at N-4 instead of the Boc group, and similar electronic properties of the substituents at the phenyl ring in the urea function were needed for the activity.The replacement of the aryl by the adamantyl group in the urea function led to compound 41 having a promising activity/selectivity profile.
From this evaluation, six compounds could be considered an interesting starting point to go deeper into this scaffold, searching for novel small molecules against anti-breast cancer based on the piperazine ring.They were selected based on their activity/security data (27, 28, 31, and 41 with IC 50 < 20 µM; 26 and 32 with IC 50 < 10 µM).
The compounds 31, 32, 35, and 37, the most selective compounds against breast cancer cells, were also tested in non-small cell lung cancer cells.All compounds decreased the viability of A549 lung cancer cells, which was measured using two different cell viability techniques (MTT and resazurin assays).Two of them, 35 and 37, showed selective anticancer activity, being less toxic to nonmalignant cells (MRC-5 and HaCaT cells).
Future research will be focused on preserving this general backbone (2-methyl or phenyl piperazine as central core) and introducing chemical diversity not only through the use of different urea and acyl groups but also different organic functions as linkers to generate different analogs with improving biological profiles.It will also study whether these compounds are selective against other types of cancer.

Figure 1 .
Figure 1.Structures of anticancer drugs containing piperazine ring highlighted in red.

Figure 1 .
Figure 1.Structures of anticancer drugs containing piperazine ring highlighted in red.

Figure 2 .
Figure 2. General structures of piperazine-derived compounds 1-3 showing acyl group and piperazine moiety in blue and red colors, respectively.

Figure 3 .
Figure 3. Structures of biaryl-urea kinase inhibitors with the urea function highlighted in green.

Figure 2 .
Figure 2. General structures of piperazine-derived compounds 1-3 showing acyl group and piperazine moiety in blue and red colors, respectively.

Figure 2 .
Figure 2. General structures of piperazine-derived compounds 1-3 showing acyl group and piperazine moiety in blue and red colors, respectively.

Figure 3 .
Figure 3. Structures of biaryl-urea kinase inhibitors with the urea function highlighted in green.

Figure 3 .
Figure 3. Structures of biaryl-urea kinase inhibitors with the urea function highlighted in green.

Figure 5 .
Figure 5. Evaluation of cytotoxic activity of compound 9 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compound for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SE) from at least two independent experiments.

Figure 5 .
Figure 5. Evaluation of cytotoxic activity of compound 9 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compound for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SE) from at least two independent experiments.

Figure 6 .
Figure 6.Evaluation of cytotoxic activity of compounds 18-20 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05.

Figure 7 .
Figure 7. Evaluation of cytotoxic activity of compounds 26, 31, and 32 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05, *** indicates p ˂ 0.001.

Figure 8 .
Figure 8. Evaluation of cytotoxic activity of compounds 35, 37, and docetaxel on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05.

Figure 6 . 19 Figure 6 .
Figure 6.Evaluation of cytotoxic activity of compounds 18-20 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p < 0.05.

Figure 7 .
Figure 7. Evaluation of cytotoxic activity of compounds 26, 31, and 32 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05, *** indicates p ˂ 0.001.

Figure 8 .
Figure 8. Evaluation of cytotoxic activity of compounds 35, 37, and docetaxel on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05.

Figure 7 . 19 Figure 6 .
Figure 7. Evaluation of cytotoxic activity of compounds 26, 31, and 32 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p < 0.05, *** indicates p < 0.001.

Figure 7 .
Figure 7. Evaluation of cytotoxic activity of compounds 26, 31, and 32 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05, *** indicates p ˂ 0.001.

Figure 8 .
Figure 8. Evaluation of cytotoxic activity of compounds 35, 37, and docetaxel on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05.

Figure 8 .
Figure 8. Evaluation of cytotoxic activity of compounds 35, 37, and docetaxel on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p < 0.05.

Figure 9 .
Figure 9. Evaluation of cytotoxic activity of compound 41 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p ˂ 0.05.

Figure 9 .
Figure 9. Evaluation of cytotoxic activity of compound 41 on human nonmalignant breast cells (MCF 10A) and human breast cancer cells (MCF7).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MCF 10A and MCF7.* indicates p < 0.05.

Figure 10 .
Figure 10.Evaluation of cytotoxic activity of compounds 31, 32, 35, 37, and cisplatin on human lung nonmalignant cells (MCR-5) and human lung cancer cells (A549).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MRC-5 and A549.* indicates p ˂ 0.05.

Figure 10 .
Figure 10.Evaluation of cytotoxic activity of compounds 31, 32, 35, 37, and cisplatin on human lung nonmalignant cells (MCR-5) and human lung cancer cells (A549).Cells were exposed to several concentrations of compounds for 72 h, and cell viability was determined with the MTT assay.Data represent mean ± SEM from at least two independent experiments.Student's t-test was performed to compare the cytotoxicity of a particular concentration of the compound between MRC-5 and A549.* indicates p < 0.05.

Figure 11 .
Figure11.Evaluation of cytotoxic activity of compounds 31, 32, 35, 37, and gemcitabine on human nonmalignant cells (HaCaT) and human cancer cells (A549).Cells were exposed to several concentrations of compounds for 72 h ("72 h T", continuous lines), and cell viability was determined with the resazurin assay.Subsequently, the cells were washed and were allowed to grow in drug-free medium for another 72 h ("72 h T + 72 h R", discontinue lines).Cell viability was again measured with the resazurin assay.Data represent mean ± SEM from at least three independent experiments.

Table 3 .
IC50 values for compounds